Detection device and methods of use

ABSTRACT

An imaging system for exciting and measuring fluorescence on or in samples comprising fluorescent materials (e.g. fluorescent labels, dyes or pigments). In one embodiment, a device is used to detect fluorescent labels on nucleic acid. In a preferred embodiment, the device is configured such that fluorescent labels in a plurality of different DNA templates are simultaneously detected.

FIELD OF INVENTION

The present invention is related to devices, methods for making devicesand methods of using devices, including devices for detectingfluorescence. In one embodiment, the present invention contemplates anoptical system, for exciting and measuring fluorescence on or in samplescomprising fluorescent materials (e.g. fluorescent labels, dyes orpigments). In one embodiment, a device is used to detect fluorescentlabels on nucleic acid. In a preferred embodiment, the device isconfigured such that fluorescent labels in a plurality of different DNAtemplates are simultaneously detected.

BACKGROUND

Scanning light microscopes have been known for several decades. Theirfunctional principal is based on a light beam being concentrated to asmall point of light (the first focal point) on a sample. The sample andthis point of light are mutually moved in such a way that a specificarea of the sample is scanned by the point of light. The light whichpenetrates the sample or is reflected by it and/or the fluorescencetriggered on or in the sample during the scanning is therefore referredto as “light originating from the sample” and is measured by one or morephotodetectors. An enlarged image is produced in that an originalmeasurement point is assigned a specific area on an image of the sample.In principle, such a scanning light microscope therefore includes: alight source, such as a laser, which produces a light beam; a sampleholder for holding the sample; an optic for producing a first focalpoint on the sample; an optical arrangement for imaging a second focalpoint using the light which shines through the sample and/or isreflected by the sample and/or which represents fluorescence triggeredon or in the sample; a photodetector for measuring the intensity of thesecond focal point; and a scanning mechanism for mutual movement of thesample and first focal point.

The approach has a number of disadvantages. First, the small focal pointmeans that only a very small portion of the sample can be addressed atone time. Second, the necessity for moving the light creates significantengineering issues and increased cost.

SUMMARY OF THE INVENTION

The present invention is related to devices, methods for making devicesand methods of using devices, including devices for detectingfluorescence. In one embodiment, the present invention contemplates anoptical system, for exciting and measuring fluorescence on or in samplescomprising fluorescent materials (e.g. fluorescent labels, dyes orpigments). In one embodiment, a device is used to detect fluorescentlabels on nucleic acid.

In a preferred embodiment, the device is configured such thatfluorescent labels in a plurality of different DNA templates aresimultaneously detected. In other words, rather than using a lightsource which creates a small focal point (such as a laser), thepreferred light source of the present invention (preferably a non-lasinglight source) illuminates a large area of a sample (e.g. at least 10% ofthe area defined by a conventional microscope slide, more preferablygreater than 20% of the area defined by a conventional microscope slide,still more preferably, greater than 50% of the area defined by aconventional microscope slide, still more preferably, greater than 70%of the area defined by a conventional microscope slide). In anotherembodiment, the preferred light source of the present invention(preferably a non-lasing light source) illuminates a defined area of achip (e.g. at least 10% of the area of the chip or 14.9×10 mm field ofview). In still another embodiment, the preferred light source of thepresent invention illuminates a larger area of a chip (e.g. up to andincluding an image area of 22 mm×22 mm, and more preferably, 22 mm×66mm). In a preferred embodiment, the system includes a light collectionmeans such as a digital camera which is capable of capturing images(capable of recording 120 um features, and more preferably, 10 micronfeatures or less).

With conventional devices, moreover, it is also difficult to performconcurrent measurements of a number of different fluorescent labels thatmay be present in a sample (or in different samples). There may bemultiple fluorescent labeling agents that have different excitationand/or emission wavelengths. Existing fluorometers, however, do notfacilitate such multiple-label experiments. Many fluorometers aredesigned for a single combination of excitation and emissionwavelengths. By contrast, in a preferred embodiment, the imaging systemof the present invention is designed for multiple excitation andemission wavelengths.

In one embodiment, the present invention contemplates an imaging system,comprising: a non-lasing light source configured such that the emittedlight from said source illuminates (and preferably converges on) a flowcell (or portion thereof), said emitted light suitable for causingvisible fluorescence of fluorescent compounds; a lens positioned tocollect at least a portion of said visible fluorescence; and a lightcollection means (e.g. a light imaging/recording means such as a chargecoupled device, a CMOS device, or other type of cameras) positioned suchthat said portion of said visible fluorescence collected by said lenspasses through toward the light collection means. In one embodiment, theimaging system is conveniently contained within a housing (portions ofwhich may be opaque or transparent). In one embodiment, the flow cell ismounted on a platform or other support structure. In another embodiment,the flow cell is attached to said housing (e.g. to a wall of thehousing, or to a mount which is attached to the housing).

The various embodiments of the imaging system of the present inventioncan be complemented with hardware (e.g. a computer) or with software.Thus, in one embodiment, the imaging system further comprises aprocessor in communication with said light collection device (e.g. CCDor other digital camera), said processor capable of recording and(optionally) optimizing images from said system. With respect tooptimizing, it may be practical and convenient to carry out optimizationof the image noise in addition to the compensation of the brightness ofthe individual partial images. Corresponding methods for adaptive,noise-optimized filtering are known, for example, from the text ofWilliam A. Pratt entitled “Digital Imaging Processing”, 1978, John Wiley& Sons, Inc., New York.

It is not intended that the present invention be limited by thearrangement of the imaging system. In one embodiment, the flow cell ison the bottom of the system and the other elements are positioned aboveit. In another embodiment, the flow cell is positioned to one side ofthe other elements, with the other elements positioned in a train ortrain-like manner. In one embodiment, the flow cell can be considered tooccupy two spatial axes X and Y, with at least some of the otherelements (e.g. the light source) positioned in the Z axis to illuminatethe flow cell (or sample therein). On the other hand, the light sourcecan be positioned differently, with the emitted light directed bymirrors into the Z axis. In one embodiment, it has been found convenientto position the flow cell such that the draining of the flow cell (e.g.the removal of fluids, such as solutions containing reagents, or washbuffers and the like) is achieved in part by gravity.

In a preferred embodiment, the flow cell is connected to a fluidicssystem, comprising various reagent and solution reservoirs in fluidiccommunication with said flow cell (e.g. via tubing). The fluidic system,in one embodiment, is pressurized and different reagents and solutionsare introduced by controlled valving (described in more detail below).In some embodiment, said flow cell comprises one or more tubingconnection ports.

It is not intended that the present invention be limited to the natureof the fluorescent compound(s) detected. The devices and systems of thepresent invention can be utilized with a variety of compounds, includingbut not limited to, dyes, inorganic molecules, multi-molecular mixturesof organic and/or inorganic molecules, crystals, heteropolymers, and thelike. For example, CdSe—CdS core-shell nanocrystals enclosed in a silicashell may be easily derivatized for coupling to a biological molecule(Bruchez et al. (1998) Science, 281: 2013 2016). Similarly, highlyfluorescent quantum dots (zinc sulfide-capped cadmium selenide) havebeen covalently coupled to biomolecules for use in ultrasensitivebiological detection (Warren and Nie (1998) Science, 281: 2016 2018).Fluorescent oligonucleotides (primers or probes) containing base-linkedor terminally-linked fluors and quenchers are well-known in the art.They can be obtained, for example, from Life Technologies (Gaithersburg,Md.), Sigma-Genosys (The Woodlands, Tex.), Genset Corp. (La Jolla,Calif.), or Synthetic Genetics (San Diego, Calif.). One of skill in theart will recognize that a large number of different fluorophores areavailable, including from commercial sources such as Molecular Probes,Eugene, Oreg. and other fluorophores are known to those of skill in theart. Useful fluorophores include: fluorescein, fluoresceinisothiocyanate (FITC), carboxy tetrachloro fluorescein (TET),NHS-fluorescein, 5 and/or 6-carboxy fluorescein (FAM), 5- (or 6-)iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),and other fluorescein derivatives, rhodamine, Lissamine rhodamine Bsulfonyl chloride, Texas red sulfonyl chloride, 5 and/or 6 carboxyrhodamine (ROX) and other rhodamine derivatives, coumarin,7-amino-methylcoumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA),and other coumarin derivatives, BODIPY™ fluorophores, Cascade Blue™fluorophores such as 8-methoxypyrene-1,3,6-trisulfonic acid trisodiumsalt, Lucifer yellow fluorophores such as3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins derivatives,Alexa fluor dyes (available from Molecular Probes, Eugene, Oreg.) andother fluorophores known to those of skill in the art. For a generallisting of useful fluorophores, see also Hermanson, G. T., BIOCONJUGATETECHNIQUES (Academic Press, San Diego, 1996). All such fluorescentmaterials are contemplated in the context of the present invention.

In the preferred embodiment of the imaging system of the presentinvention, said flow cell comprises an array of nucleic acid (e.g. thearray is contained within the flow cell), at least a portion of saidnucleic acid comprising fluorescent dyes (e.g. fluorescent labelscovalently attached to a nucleotide incorporated in said nucleic acid).Preferably, said flow cell comprises means for introducing reagents insolution (such that biological reactions can take place on or in thearray), said reagents selected from the group consisting of labelednucleotides and enzymes (typically introduced in solution, such asbuffers; the buffers also being useful alone for washing the array freeof reactants).

It is not intended that the present invention be limited by the natureof the non-lasing light source. A variety of non-laser type lightsources are contemplated, including but not limited to light emittingdiodes (LEDs). In a preferred embodiment, the present inventioncontemplates a imaging system, wherein said non-lasing light sourcecomprises a plurality of light emitting diodes. In a particularlypreferred embodiment, said plurality of light emitting diodes comprisesfour different sets of light emitting diodes, each of which emit adifferent wavelength of light (e.g. 488 nm, 530 nm, 585 nm, and 615 nm).The light emitting diodes can be configured in an array (e.g. linear orcircular) such that the emitting light illuminates (and preferablyconverges on) a sample (e.g. material on a microscope slide, an array,an array contained within a flow cell, a flow cell, etc.). It is notintended that the present invention be limited by the number of lightemitting diodes. In one embodiment, the present invention contemplatesthe simple case where just four different LEDs are used (as distinctfrom four different sets of LEDs), each emitting a different wavelength.Even where four different sets are used, the present inventioncontemplates embodiments wherein there are equal numbers within eachset, and embodiments where some or all sets have different numbers oflight emitting diodes. Thus, for example, in a circular array of 20LEDs, 7 may emit at one particular wavelength, while 3 may emit atanother, with the remaining 10 comprising two sets of 5 LEDs, each setemitting at yet other wavelengths. Optionally, in order to further limitor narrow the wavelengths emitted by the LEDs, they may be combined withnarrow bandpass filters placed between the LEDs and the sample (e.g. theflow cell containing the array on a chip). Further embodiments mayoptionally include additional elements used to shape the light (e.g. ashaping lens and/or collimating lens) from the light source.

In a preferred embodiment, the imaging system comprises filterspositioned in front of the lens, within the lens, or between the lensand the light collection means. Preferably, the filters are opticalbandpass filters which can be positioned in a linear or circular manner.In a particularly preferred embodiment of the imaging system describedabove, the system further comprises a filter wheel comprising a hub anda plurality of radially extending mounts, each of said mounts containingan optical bandpass filter. In a preferred embodiment, four such filtersare employed, each selected for different preferred wavelengths. In oneembodiment, four 50 mm interference filters are employed to allow themeasurement of the fluorescent emissions of four different fluorophores.

The filters can be stationary or can be movable. In a preferredembodiment of the imaging system described above, the system furthercomprises a motor engaged (either directly or through transmissionelements) with said hub of said filter wheel, wherein the motor isadapted to rotate said filter wheel to position any one of the pluralityof filters between the light collection means (e.g. a charge coupleddevice) and the sample (e.g. the flow cell). Other means of limiting thebandwidth of light such as dichroic mirrors may also be used as a kindof filter.

In another embodiment, the present invention contemplates an imagingsystem, comprising: an array of light emitting diodes configured suchthat the emitted light illuminates (and preferably converges on) asample comprising fluorescent materials, said emitted light suitable forcausing visible fluorescence of fluorescent materials; a lens positionedto collect at least a portion of said visible fluorescence; and a chargecoupled device positioned such that said portion of said visiblefluorescence collected by said lens passes through toward the chargecoupled device. In a preferred embodiment of the imaging system, saidarray of light emitting diodes comprises four different light emittingdiode sets, each of which emit a different wavelength of light (e.g. 488nm, 530 nm, 585 nm, and 615 nm). The light emitting diodes can beconfigured in an array (e.g. linear or circular) such that the emittinglight illuminates (and preferably converges on) a sample (e.g. materialon a microscope slide, an array, an array contained within a flow cell,a flow cell, etc.). It is not intended that the present invention belimited by the number of light emitting diodes. Even where fourdifferent sets are used, the present invention contemplates embodimentswherein there are equal numbers within each set, and embodiments wheresome or all sets have different numbers of light emitting diodes. Thus,for example, in a circular array of 20 LEDs, 7 may emit at oneparticular wavelength, while 3 may emit at another, with the remaining10 comprising two sets of 5 LEDs, each set emitting at yet otherwavelengths. Optionally, in order to further limit or narrow thewavelengths emitted by the LEDs, they may be combined with narrowbandpass filters placed between the LEDs and the sample (e.g. the flowcell containing the array on a chip). Further embodiments may optionallyinclude additional elements used to shape the light (e.g. a shaping lensand/or collimating lens) from the light source.

In one embodiment of the imaging system, said sample comprises nucleicacid, at least a portion of said nucleic acid comprising fluorescentdyes. In a preferred embodiment, said sample is contained within a flowcell. In one embodiment, said flow cell comprises means for introducingreagents (typically in solution to said sample). In one embodiment, saidreagents are selected from the group consisting of labeled nucleotidesand enzymes (e.g. polymerases). As discussed above, in one embodiment,the flow cell is in fluidic communication with a fluidics system (viatubing and connection ports).

In a preferred embodiment, the imaging system comprises filterspositioned in front of the lens, within the lens, or between the lensand the light collection means. Preferably, the filters are opticalbandpass filters which can be positioned in a linear or circular manner.In a particularly preferred embodiment of the imaging system describedabove, the system further comprises a filter wheel comprising a hub anda plurality of radially extending mounts, each of said mounts containingan optical bandpass filter. In a preferred embodiment, four such filtersare employed, each selected for different preferred wavelengths. In oneembodiment, four 50 mm interference filters are employed to allow themeasurement of the fluorescent emissions of four different fluorophores.

The filters can be stationary or can be movable. In a preferredembodiment of the imaging system described above, the system furthercomprises a motor engaged (either directly or through transmissionelements) with said hub of said filter wheel, wherein the motor isadapted to rotate said filter wheel to position any one of the pluralityof filters between the light collection means (e.g. a charge coupleddevice) and the sample (e.g. the flow cell).

In one embodiment, the present invention contemplates manufacturing animaging system, comprising assembling: a non-lasing light sourceconfigured such that the emitted light from said source illuminates (andpreferably converges on) a flow cell (or portion thereof), said emittedlight suitable for causing visible fluorescence of fluorescentcompounds; a lens positioned to collect at least a portion of saidvisible fluorescence; and a light collection means (e.g. a chargecoupled device, a CMOS device, or other type of cameras) positioned suchthat said portion of said visible fluorescence collected by said lenspasses through toward the light collection means. In one embodiment,said light source comprises LEDs (e.g. a circular array of LEDs).

In one embodiment, the present invention contemplates a methodcomprising: a) providing an imaging system, said imaging systemcomprising a non-lasing light source configured such that the emittedlight from said source illuminates (and preferably converges on) a flowcell (or portion thereof) comprising an array of biomolecules, saidemitted light suitable for causing visible fluorescence of fluorescentcompounds; a lens positioned to collect at least a portion of saidvisible fluorescence; and a light collection means (e.g. a chargecoupled device, a CMOS device, or other type of camera) positioned suchthat said portion of said visible fluorescence collected by said lenspasses through toward the light collection means; b) introducing asolution into said flow cell, said solution comprising one or morefluorescent compounds, under conditions such that at least a portion ofsaid fluorescent compounds attaches to at least a portion of said arrayof biomolecules, so as to create treated biomolecules, and c) imagingsaid treated biomolecules with said imaging system. In one embodiment ofthis method, the biomolecules comprises nucleic acid. In one embodimentof this method, the solution comprises oligonucleotides comprisingfluorescent tags, wherein a portion of said oligonucleotides hybridizewith a portion of said nucleic acid biomolecules of said array.

In another embodiment, said biomolecules comprise nucleic acid and saidsolution comprises fluorescently-labeled nucleotides and an enzymecapable of causing at least a portion of said nucleotides to beincorporated into at least a portion of said nucleic acid biomoleculesof said array. In one embodiment, said nucleotides are BODIPY-labelednucleotides. In another embodiment, a second solution is employedcomprising one or more enzymes (or chemicals) capable of removing saidfluorescent labels. In one embodiment, first and second solutions areused stepwise whereby labels are introduced, imaged, and subsequentlyremoved (the cycle being repeated two times, more preferably 10 times ormore).

In another embodiment, the present invention contemplates a method,comprising a) constructing a crosstalk matrix from measurement of puredyes, b) inverting the matrix and c) using it to separate subsequentmeasurements using imaging system (such as the LED illumination-baseddetector system describe above). This crosstalk matrix can beconstructed for a four color system (but is not limited to four colors).

DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows one embodiment of the imaging system of thepresent invention, said embodiment comprising a) a circular array ofLEDs configured such that the emitted light converges on a region orplatform (e.g. a position for a sample, flow cell, etc.) so as to excitefluorescence of fluorescent material, b) a lens assembly positionedabove the region so as to capture at least a portion of saidfluorescence, c) a filter wheel comprising bandpass filters, and d)light collection means (in this case a cooled CCD camera), wherein saidfilter wheel is positioned between the region where the light convergesand the light collection means.

FIGS. 2A-B schematically show one embodiment of a flow cell. FIG. 2Ashows a three dimensional translucent view of a flow cell, comprisingfluid tubing connections, cartridge heaters, and O-ring seal. FIG. 2B isa two dimensional drawing of a side view of a flow cell, showing anarray or slide with spaced spots on the surface (representing positionsfor biomolecules and/or anchoring molecules), said array positioned in afluid channel such that solutions of buffers and/or reagents can beintroduced over the surface under conditions whereby reactions and/orwashing can be achieved. The arrows show one preferred direction offluid flow, with entrance and exit ports, as well as one preferredmethod of sealing (O-ring seal).

FIG. 3 schematically shows one embodiment of a fluidics system,comprising a variety of illustrative reagent and buffer reservoirs incommunication (via tubing or other channeling into a manifold comprisingvalves) with one embodiment of a flow cell (comprising a side entranceport and one or more heaters), wherein the array or chip is inverted andthe exit port is on the bottom, thereby permitting the fluid channel tobe drained at least in part by gravity so that waste can be readilycollected into a reservoir.

FIG. 4A schematically shows another embodiment of an imaging system,wherein two flow cells and two cameras are employed to increase capacityand efficiency (e.g. while one chip in a first flow cell is undergoingone or more reaction steps, a second chip in a second flow cell is beingscanned and imaged). FIG. 4B shows a closer illustration of the threepiece lens system, including the two pair color filters and dichroicbeam splitters.

FIGS. 5A-5D show an illustrative excitation and emission filterselection (grey rectangles) for four illustrative dyes, relative to thedye's excitation (dashed) and emission (solid) spectra. FIG. 5A showsthe excitation and emission filter selection for the dye BODIPY FL. FIG.5B shows the excitation and emission filter selection for the dye R6G.FIG. 5C shows the excitation and emission filter selection for the dyeROX. FIG. 5D shows the excitation and emission filter selection for thedye BODIPY 650.

FIGS. 6A-6B show the raw data (FIG. 6A) and crosstalk adjusted data(FIG. 6B) for four illustrative dyes.

DETAILED DESCRIPTION

The present invention contemplates a fluorescent detection system and aflow cell for processing biomolecules (e.g. nucleic acid samples)arrayed on a “chip” or other surface (e.g. microscope slide, etc.). Theflow cell permits the user to perform biological reactions, includingbut not limited to, hybridization and sequencing of nucleic acids.

It is not intended that the present invention be limited to particularlight sources. By way of example only, the system can employultra-bright LEDs (such as those available from Philips LumiledsLighting Co., San Jose, Calif.) of different colors to excite dyesattached to the arrayed nucleic acids. These LEDs are more costeffective and longer life than conventionally used gas or solid statelasers. Other non-lasing sources of lights such as incandescent orfluorescent lamps may also be used.

FIG. 1 shows a useful configuration of the LEDs, whereby the emittedlight converges on a region or platform (e.g. suitable for positioningthe flow cell or sample). However, linear arrays of LEDs can also beused.

It is not intended that the present invention be limited to particularlight collection devices. By way of example only, the system may employa high sensitivity CCD camera (such as those available from RoperScientific, Inc., Photometric division, Tucson Ariz. or those availablefrom Apogee Instruments, Roseville, Calif.) to image the fluorescentdyes and make measurements of their intensity. The CCD cameras may alsobe cooled to increase their sensitivity to low noise level signals.These may also be CMOS, vidicon or other types of electronic camerasystems.

Since LED illumination light is not a collimated beam as from lasers, itis therefore an appropriate choice for imaging a larger area of manynucleic acid spots. To get sufficient light and therefore fluorescentsignals over the larger area, the area seen by each pixel of the cameramust be of sufficient size to allow enough fluorescent dye molecules tocreate a sufficient signal (for example, an Apogee U13 CCD available has1.3 megapixels of 16 microns in size, while the Apogee U32 has 3.2megapixels of 6.8 microns in size).

To increase capacity and efficiency, the present invention contemplatesin one embodiment, a two flow cell system (e.g. while one chip in afirst flow cell is undergoing one or more reaction steps, a second chipin a second flow cell is being scanned and imaged) with a single camera.In yet another embodiment of an imaging system, two flow cells and twocameras are employed (FIG. 4A and shown as a close up in FIG. 4B).

In one embodiment, the chip containing the array of nucleic acid spotsis processed in a transparent flow cell incorporated within theinstrument, which flows reagent past the spots and produces the signalsrequired for sequencing (see FIGS. 2A and 2B). In a preferredembodiment, the chip remains in the flow cell while it is imaged by theLED detector. The flow cell and associated reagents adds the nucleicacids, enzymes, buffers, etc. that are required to produce thefluorescent signals required for each sequencing step, then the flowcell delivered the required reagents to remove the fluorescent signalsin preparation for the next cycle. Measurement by the detector occursbetween these two steps. In order for reactions to take place, the flowchannels need to be of sufficient dimensions. For example, the channelby the array should be at least 0.1 mm in depth (more preferably 0.5 mmin depth) and the volume formed by the chip, the block and the sealshould be at least 100 microliters in volume (more preferably, between100 and 700 microliters, and still more preferably, between 150 and 300microliters, e.g. 200 microliters, in volume).

The flow cell is preferably motionless (i.e. not moved during reactionsor imaging). On the other hand, the flow cell can readily be mounted ona rotary or one or more linear stages, permitting movement. For example,in a two flow cell embodiment, the two flow cells may move up and down(or side to side) across the imaging system. Movement may be desiredwhere additional processes are desired (e.g. where exposure to UV lightis desired for photochemical reactions within the flow cell, such asremoval of photocleavable fluorescent labels), when multiple flow cellsshare a single camera, or when the field of view of the detection systemis smaller than the desired area to be measured on the flow cell. Thedetector system may also be moved instead of the flow cell.

The flow cell is preferably in fluid communication with a fluidicssystem (see illustrative system shown in FIG. 3. In one embodiment, eachbottle is pressurized with a small positive gas pressure. Opening theappropriate valve allows reagent to flow from the source bottle throughthe flow cell to the appropriate collection vessel(s). In oneembodiment, the nucleotides and polymerase solutions will be recoveredin a separate collection bottle for re-use in a subsequent cycle. In oneembodiment, hazardous waste will be recovered in a separate collectionbottle. The bottle and valve configuration allow the wash fluid to flushthe entire valve train for the system as well as the flow cell. In oneembodiment, the process steps comprise: 1) flushing the system with washreagent, 2) introducing nucleotides (e.g. flowing a nucleotide cocktail)and polymerase, 3) flushing the system with wash reagent, 4) introducingde-blocking reagent (enzyme or compounds capable of removing protectivegroups in order to permit nucleic acid extension by a polymerase), 5)image, 6) introduce label removing reagent (enzyme or compounds capableof removing fluorescent labels), and 7) flushing the system with washreagent.

The system can be made to include a user interface system. The Labview(National Instruments, Austin, Tex.) system is available and providesrelatively simply software for computer controlled systems. Galil MotionControl (Rocklin, Calif.) provides motion control systems that can beinterfaced to control the instrument.

Example Method for Removing Crosstalk Between Detected FluorescentSignals for a Multicolor System

Previous sequencing systems utilizing lasers have attempted to minimizethe number of lasers in order to reduce costs (for example ABI Prismsequencers). For a four color detection system using LEDs, the lightsources are fairly inexpensive and it is desirable to have four separatecolor light sources in order to reduce crosstalk between colors asfollows.

To determine actual fluorescent intensities for the four colors, A, B, Cand D from measured detector outputs, M_(A), M_(B), M_(C), M_(D) incorresponding channels, you need to know all of the crosstalk factors:R_(AB), R_(BA), R_(BC), R_(CB), R_(CD), R_(DC). Six crosstalk factorsare used for illustrative purposes. There may be more or fewer factorswhich may be incorporated into the analysis.

For example, R_(AB) is the ratio between the portion of the signal inthe A channel coming from the B dye and the actual intensity of the Bdye. If for instance R_(AB) is 20%, then the A channel will have anadditional signal equal to 0.2 times the actual B dye intensity in the Bchannel. Thus for channel B, the observed measurement, M_(B), is thedirect measurement of B and the two contributions from the adjacentchannels (if any):M _(B) =B+R _(BA) A+R _(BC) C  (1)For the four channels, this may be written in matrix form:

$\begin{matrix}{\begin{bmatrix}M_{A} \\M_{B} \\M_{C} \\M_{D}\end{bmatrix} = {{{K\begin{bmatrix}A \\B \\C \\D\end{bmatrix}}{\mspace{11mu}\;}{where}\mspace{14mu} K} = {\begin{bmatrix}1 & R_{AB} & 0 & 0 \\R_{BA} & 1 & R_{BC} & 0 \\0 & R_{CB} & 1 & R_{CD} \\0 & 0 & R_{DC} & 1\end{bmatrix}.}}} & (2)\end{matrix}$Each of the six crosstalk factors may be determined through a simpleexperiment with pure dyes. Some may be zero and they might vary withintensity, so we may need a table of a number of values for eachdepending on the measured intensity range. We want to solve for theactual fluorescent signals, A, B, C and D given the detectormeasurements, M_(A), M_(B), M_(C), M_(D). Thus, we want to solve theabove matrix equation (2). This is:

$\begin{matrix}{\begin{bmatrix}A \\B \\C \\D\end{bmatrix}\; = {K^{- 1}\begin{bmatrix}M_{A} \\M_{B} \\M_{C} \\M_{D}\end{bmatrix}}} & (3)\end{matrix}$where K⁻¹ is the inverse of matrix K. Although this may be written outin terms of the six crosstalk factors, it is somewhat complex and isbest performed by plugging in the numbers and letting the computer takethe inverse. FIG. 6 shows the raw data (6A) and crosstalk adjusted data(6B) for four illustrative dyes.

The invention claimed is:
 1. A method comprising: a) providing animaging system, said imaging system comprising i) a non-lasing lightsource, emitting different wavelengths of light, configured such thatthe emitted visible light from said source illuminates a portion of aflow cell, ii) said flow cell comprising an array of biomolecules on asurface, wherein said emitted light illuminates said surface, whereinsaid array faces inwardly so that reagents introduced in solution cancontact said biomolecules, wherein said flow cell comprises a fluidicconnection to at least one reagent bottle wherein said at least onereagent bottle is under a positive pressure, iii) a lens positioned tocollect a portion of a visible fluorescence; and iv) a camera; b)introducing a solution via said fluidic connection into said flow cell,said solution comprising a plurality of fluorescent compounds havingdifferent excitation and emission wavelengths, under conditions suchthat a portion of said fluorescent compounds attaches to a portion ofsaid array of biomolecules, so as to create treated biomolecules, c)illuminating said flow cell with said light source emitting differentwavelengths of light, wherein said emitted visible light from saidsource converges on said fluorescent compounds, and d) concurrentlyimaging said treated biomolecules illuminated with said light sourceemitting different wavelengths of light with said imaging system.
 2. Themethod of claim 1, wherein said flow cell is transparent.
 3. The methodof claim 1, wherein said array of biomolecules comprises an array ofnucleic acid.
 4. The method of claim 1, wherein said fluorescentcompounds comprise labeled nucleotides.
 5. The method of claim 4,wherein said solution of step b) further comprises polymerase.
 6. Themethod of claim 1, further comprising, prior to step d) removing saidsolution from said flow cell.
 7. The method of claim 6, wherein saidremoving is achieved in part by gravity.
 8. The method of claim 1,wherein said flow cell is connected to a fluidics system.
 9. The methodof claim 1, wherein said solution is under positive pressure and isintroduced at step b) by controlled valving.
 10. A method comprising: a)providing an imaging system, said imaging system comprising i) first,second, third and fourth non-lasing light sources, each source emittinga different wavelength of visible light and configured such that theemitted light from said source illuminates a portion of a flow cell, ii)said flow cell comprising an array of biomolecules positioned so thatreagents introduced in solution can contact said biomolecules, whereinsaid flow cell comprises a fluidic connection to at least one reagentbottle wherein said at least one reagent bottle is under a positivepressure, iii) a lens positioned to collect a portion of a visiblefluorescence; and iv) a camera; b) introducing a solution via saidfluidic connection into said flow cell, said solution comprising aplurality of fluorescent compounds having different excitation andemission wavelengths, under conditions such that a portion of saidfluorescent compounds attaches to a portion of said array ofbiomolecules, so as to create treated biomolecules, c) illuminating saidflow cell with said light source emitting different wavelengths oflight, and d) concurrently imaging said treated biomolecules illuminatedwith said light source emitting different wavelengths of light with saidimaging system.
 11. The method of claim 10, wherein said light sourcesare light emitting diodes.
 12. The method of claim 10, wherein two ofsaid four wavelengths comprise 488 nm and 530 nm.
 13. The method ofclaim 10, wherein said flow cell comprises one or more flow channels,and said array of biomolecules is positioned in a channel underconditions such that said array contacts said solution upon saidintroducing of said solution of step (b).
 14. The method of claim 12,wherein one of said one or more flow channels has a first surface and asecond surface, and wherein said array is positioned on said firstsurface.
 15. A method comprising: a) providing an imaging system, saidimaging system comprising i) first and second non-lasing light sources,each source emitting different wavelengths of visible light andconfigured such that the emitted light from said source illuminates aportion of a flow cell, ii) said flow cell comprising biomoleculespositioned so that reagents introduced in solution can contact saidbiomolecules, wherein said array of biomolecules comprises an array ofnucleic acid, wherein said flow cell comprises a fluidic connection toat least one reagent bottle wherein said at least one reagent bottle isunder a positive pressure, iii) a lens positioned to collect a portionof a visible fluorescence; and iv) a camera; b) introducing a solutionvia said fluidic connection into said flow cell, said solutioncomprising a plurality of fluorescent compounds having differentexcitation and emission wavelengths, under conditions such that aportion of said fluorescent compounds attaches to a portion of saidbiomolecules, so as to create treated biomolecules, c) illuminating saidflow cell with said light source emitting different wavelengths oflight, wherein said emitted visible light from said source converges onsaid fluorescent compounds, and d) concurrently imaging said treatedbiomolecules illuminated with said light source emitting differentwavelengths of light with said imaging system.
 16. The method of claim15, wherein said light sources are light emitting diodes.